Stimulation of Vascular Endothelial Growth Factor Gene Transcription by all trans Retinoic Acid through Sp1 and Sp3 Sites in Human Bronchioloalveolar Carcinoma Cells

Abstract

In this study, we examined the effects of all trans-retinoic acid (at-RA) on the vascular endothelial growth factor (VEGF) expression in human bronchioloalveolar carcinoma NCI-H322 cells to evaluate the potential of at-RA to affect tumor progression. Northern blot and enzyme-linked immunosorbent assay analyses indicate that VEGF production is significantly increased by 1 microM of at-RA. A series of 5'-deletion and site-directed mutation analyses indicated that G+C-rich sequence located at -81 and -52 was required for at-RA- and retinoic acid receptor alpha-mediated induction of VEGF promoter. Electrophoretic mobility shift and supershift assays showed that major constituents of nuclear factors binding to G+C-rich sequences are Sp1 and Sp3. Pretreatment with cycloheximide, a protein synthesis inhibitor, prevented the at-RA-mediated induction of VEGF mRNA expression. Likewise, at-RA-mediated VEGF expression was completely blocked in the presence of genistein, an inhibitor for tyrosine kinases. These results suggest that an increase in transcription of the VEGF promoter by at-RA is mediated through Sp1 site, and both new protein synthesis and tyrosine kinase activation are necessary for this induction. Because VEGF can promote neovascularization in cancer cells, an induction of VEGF by at-RA may preclude the therapeutic application of at-RA to cancer patients.

The transcription factor Net regulates the angiogenic switch

“…Two complexes were detected that were putatively identified as Sp1 and Sp3 from comparisons with previous reports (Fig. 5B, lane 4; Maeno et al 2002). The same two bands were also present in nuclear extracts from cells transfected with just the Net vector, just the Ras-V12 vector, or the empty vectors (Fig.…”

“…In addition, several minor bands were detected that migrated more slowly than Sp1 and that were supershifted with the Sp1 + Sp3 antibodies (Fig. 5C, indicated by dots, lanes 1-3; similar higher mobility complexes have been detected by others: Maeno et al 2002). None of the bands were supershifted by Net antibodies (Fig.…”

“…The mouse VEGF promoter contains different motifs (Shima et al 1996), including a hypoxia responsive element (HRF; Oosthuyse et al 2001) and two proximal Sp1 motifs (Maeno et al 2002). There are a number of other putative motifs; some of those in the −449/+1 region are shown in Figure 5A.…”

Retinoids Increase Lung Elastin Expression But Fail to Alter Morphology or Angiogenesis Genes in Premature Ventilated Baboons

“…Reports on the effects of retinoids on angiogenesis vary widely. Retinoic acid increases VEGF expression in lung epithelial cells (14) and stimulates fetal pulmonary microvascular endothelial cell proliferation in vitro (23). Retinoids reverse dexamethasone-mediated repression of lung VEGF receptor 2 in newborn mice (30) and restore alveolarization caused by pharmacologic blockade of this receptor (23).…”

“…Retinoid signaling is required for alveolar capillary development. Vitamin A supplementation may decrease the incidence of BPD by stimulating VEGF expression in alveolar epithelial cells (11,14) or through other proangiogenic pathways (15). Retinoid signaling also increases lung endothelial proliferation (16).…”